Simian virus 40 is a DNA tumor virus, which is associated with human mesotheliomas and pediatric brain tumors and possibly other human malignancies. Two SV40 proteins are required to transform human cells, the large-T (LT) and small-t (ST) antigens. LT is a well-studied transforming protein that interacts with tumor suppressor genes like Rb and p53. We have shown that ST is also essential for transformation of human cells and that both SV40 proteins cooperate in the absence of serum to drive cell cycle progression of density-arrested human fibroblasts. This occurs, at least in part, through regulation of cyclin-kinase inhibitors, with LT expression reducing the cki p21 while ST expression reduces p27 levels. Binding of ST to protein phosphatase 2A is necessary for this p27 reduction. The first aim of our proposed research is to determine the mechanism of p27 reduction and whether p27 reduction is sufficient to explain effects of ST on growth promotion and transformation. In aim two, we will explore the significance of the anti-apoptotic kinase, AKT, to ST function. Experiments in this aim focus primarily on mechanisms by which ST activates AKT, but other potential targets of ST will be investigated including new genes identified by comparative microarrays. An important part of this aim is to determine whether the requirement for ST in human cell transformation can be met by up- or down-regulation of a combination of cellular activities. In aim 3, will explore a newly developed model for human mesothelial cell transformation. We have established mesothelial cells in culture using telomerase and LT, but these do not exhibit transformed properties such as focus formation or anchorage independent growth until ST is introduced. We will investigate the effects of ST on established mesothelial cells and the role of specific cellular activities in this process. In addition, the drift of ST-expressing mesothelial cells toward more aneuploid populations will be evaluated with particular interest on whether ST contributes actively to this process. These approaches should provide crucial information to define the essential role of ST in transformation in human systems, and may serve as a prototype for studies of other human cell types that may be targets of SV40 tumorigenesis.